The life cycle of retroviruses is characterized by two steps, which are carried out by two virally encoded enzymes, reverse transcriptase (RT) and integrase (IN). In the first of these steps, RT converts the single-stranded viral RNAs found in virions into a linear double-stranded DNA that is longer than the RNAs from which it is derived. In the second step, integrase (IN) inserts this linear viral DNA into the host genome. Both steps are essential for the retroviral life cycle; both RT and IN are key anti-HIV drug targets. The development of new broadly effective, low-toxicity anti-HIV drugs against such targets is one of the high-priority research goals of the NIH, in part because of problems with emerging drug resistance in the developing world. ____The conversion of retroviral genomic RNA into DNA involves the two enzymatic activities of RT: a polymerase that can copy either RNA or DNA, and a ribonuclease H (RNase H) that cleaves RNA if it is part of an RNA/DNA hybrid. Although the RNase H of RT is essential for viral replication, there are no anti-HIV drugs that target RNase H. The two clinically important classes of anti-RT drugs -- nucleoside analogs (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) -- instead target the polymerase. ____A major focus of our work has been on the mechanism(s) of RT inhibitor resistance to these two classes of drugs. There is, at this point, a reasonably good understanding of the mechanism(s) of NNRTI resistance, and considerable progress has been made in understanding NRTI resistance, although some important issues remain. The NRTIs that are currently used to treat HIV 1 infections lack the 3'-OH found on normal deoxynucleosides. If an NRTI is incorporated into viral DNA by RT, polymerization is blocked. Because NRTIs can also be incorporated into the mitochondrial and nuclear DNA of host cells, these drugs can be toxic to patients, particularly because HIV drug therapy is usually lifelong. In contrast to NRTIs, NNRTIs are, as a group, relatively nontoxic, but are prone to the development of resistance. We have focused on understanding NRTI resistance and on developing NNRTIs that are more broadly effective against the known drug-resistant mutants of HIV-1. We have also recently turned our attention to the RNase H activity of HIV-1 RT, which could be an effective (and important) target for the development of new inhibitors and drugs. ____In our recent efforts to develop more effective NNRTIs, we generated analogs of the clinically approved NNRTI rilpivirine (RPV) and participated in a collaborative effort to find new ways to develop NNRTIs. RPV is relatively effective against NNRTI-resistant HIV-1 mutants, and there are promising data to suggest that it can be combined with the investigational drug cabotegravir and formulated for use as a long-acting injection/implant. All of the RPV analogs that we are continuing to study are broadly effective against a panel of 34 single, double, and triple NNRTI-resistant mutants that we use. Our best compounds are, in this regard, better than RPV. However, it may not be possible to develop an NNRTI that can overcome all of the possible resistance mutations. Therefore, we are testing an alternative strategy: can we develop (or identify) pairs of NNRTIs that are more effective (in terms of the development of resistance) than either compound used alone? Because the development of resistance is a problem, the drug combinations used in antiretroviral therapy are chosen to avoid selecting mutations that confer resistance to more than one drug. Drugs from the same class can be used together if they do not select overlapping resistance mutations. For example, FTC and TDF are NRTIs that are used together because they select different resistance mutations. ____Although most NNRTIs select for overlapping resistance mutations, we were interested in whether the new NNRTI doravirine (DOR) and RPV have a complementary susceptibility to NNRTI mutants, as Merck suggested, based on testing the compounds against a limited number of resistant mutants. We also wanted to know, if the two drugs have nonoverlapping susceptibilities to a broad panel of resistant mutants, whether we could understand why their susceptibilities differ. We tested, using a single-round HIV-1 infection assay, both RPV and DOR against our panel of 22 single, 10 double, and 2 triple NNRTI-resistant mutants. Several of our mutants displayed decreased susceptibility to DOR. However, with the possible exception of E138K, which is selected by RPV but does not, in experiments done by us or others, show much loss of susceptibility to RPV, our data suggest that the mutations that reduce the potency of DOR and RPV are nonoverlapping. Thus, we think that these two NNRTIs have the potential to be used in combination therapy. We used molecular modeling, based on the crystal structures of DOR and RPV bound to RT, to show that the different susceptibility profiles of these compounds correlate with differences in the ways the compounds bind to RT (DOR binds deeper in the pocket; RPV, nearer the outer rim). We think that our understanding of the differences in the binding interactions of these NNRTIs with RT can be used to develop pairs of compounds with nonoverlapping susceptibilities to RT mutations. ____Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and IN), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics. We recently reported analysis of three compounds that selectively inhibit the RNase H of HIV-1 RT. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RNase H active site. These compounds appear to be relatively specific; they do not inhibit either human RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to IN strand transfer inhibitors (INSTIs), NRTIs, or NNRTIs, suggesting that their ability to block HIV replication is related to their ability to block RNase H cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds._____PATENTS LINKED TO THIS PROJECT: (1) Hughes S, Boyer P, Linial M, Stenbak C, Clark P: Foamy Virus Mutant Reverse Transcriptase. U.S. Patent #7,560,117, issued July 14, 2009. (2) Vu BC, Siddiqui MA, Marquez VE, Hughes SH, Boyer PL: C4'-Substituted-2-Deoxyadenosine Analogs and Methods of Treating HIV. U.S. Patent #8,513,214, issued August 20, 2013. (3) Michejda CJ, Szekely Z, Hariprakasha HK, Hughes SH: Benzidole Derivatives and Method for Treating HIV/AIDS. Patent pending: PCT/US2007/080957 (PC application), submitted in 2006.